Human tPA production using vectors coding for DHFR protein

ABSTRACT

A method for producing tissue plasminogen activator (t-PA) in eukaryotic host cells is disclosed. Enhanced levels of t-PA production are obtained by co-amplification of the t-PA gene through treatment of cultures transformed with mutant or wild type DHFR with methotrexate.

This is a continuation of application Ser. No. 08/162,354 filed 3 Dec.1993, now U.S. Pat. No. 5,424,198 which is a division of applicationSer. No. 07/663,103, filed 28 Feb. 1991, now U.S. Pat. No. 5,268,291,which is a continuation of application Ser. No. 07/499,209, filed 22Mar. 1990, now U.S. Pat. No. 5,010,002, which is a continuation ofapplication Ser. No. 07/149,990, filed 27 Jan. 1988, abandoned, which isa continuation of application Ser. No. 06/459,153, filed 19 Jan. 1983,abandoned. Reference is hereby made under 35 USC 120/121 to U.S. Ser.No. 06/374,860 filed 5 May 1982, U.S. Ser. No. 06/398,003, filed 14 Jul.1982, and U.S. Ser. No. 06/483,052, filed 7 Apr. 1983.

BACKGROUND OF THE INVENTION

The invention herein relates to the production of human tissueplasminogen activator (tPA) in a transformant host cell culture. Morespecifically, the invention relates to vectors, cells, and methods ofproducing tPA in conjunction with expression of the sequences for codingfor dihydrofolate reductase (DHFR) protein in such cells.

The production of tPA using recombinant techniques has been disclosed inU.S. application Ser. No. 398,003, filed Jul. 14, 1982 which is acontinuation in part of U.S. Ser. No. 374,860 filed May 5, 1982; thecontents of both applications are incorporated herein by reference.These applications describe the construction of plasmids containing thecoding sequences for tPA, and describe the activity and utility of tPAso produced.

It has also been found, as set forth in applications U.S. Ser. No.06/459,152, filed 19 Jan. 1983, now U.S. Pat. No. 4,713,339, and U.S.Ser. No. 06/459,151, filed 19 Jan. 1983, now abandoned, and incorporatedherein by reference, that a DNA sequence encoding for a DHFR protein canbe utilized as a marker for transfection of a sequence coding for adesired heterologous protein in suitable host cells. The DHFR sequencecan also be used as a secondary sequence permitting control of theproduction of the desired protein. These applications disclose such ause, both of wild type DHFR, and of a mutant DHFR which is resistant tomethotrexate.

A problem frequently encountered in the production of polypeptides in aforeign host is the necessity to have some mechanism to regulate,usually to enhance, the production of the desired protein. In the caseof tPA, which forms the subject matter of this invention, a secondarycoding sequence comprising DHFR which is affected by an externallycontrolled parameter, such as methotrexate, is utilized to permitcontrol of expression by control of the methotrexate (MTX)concentration.

Methotrexate is a drug which is normally fatal to cells capable of itsuptake. However, certain cells are able to grow in the presence ofcontrolled levels of MTX. One of the several mechanisms wherebymethotrexate resistance is effected is that whereby amplification of thegene coding for the DHFR coding sequence is stimulated (Schimke, RobertT. et al, Science, 202: 1051 (1978); Biedler, J. L. et al, Cancer Res.32: 153 (1972); Chang, S. E., et al, Cell, 7: 391 (1976)).

It has further been shown that amplification of the gene for DHFR mayfurther cause amplification of associated sequences which code for otherproteins. This appears to be the case when the associated protein ishepatitis B surface antigen (HBsAg) (Christman, J. et al, Proc. Natl.Acad. Sci., 79: 1815 (1982)); the E. coli protein XGPRT (Ringold,Gordon, et al, J. Molec. and Appl. Gen., 1: 165 (1981)); and anendogenous sequence from a DHFR/SV40 plasmid combination (Kaufman, R. F.et al, J. Molec. Biol., 159: 601 (1982)).

Other mechanisms for conferring methotrexate resistance includediminution of the binding affinity of the DHFR protein, so that it isless susceptible to methotrexate (Flintoff, W. F. et al, Somat. CellGenet., 2:245 (1976)) but in this instance, amplification appears tooccur as well.

Thus it would appear that the genes both for wild type DHFR and for DHFRwhich is resistant to MTX by virtue of its own decreased bindingcapacity are amplified by the presence of MTX. Hence, in principle, theinvention herein concerns using the impact of DHFR sequenceamplification on associated protein coding sequences to provide acontrol mechanism which permits enhanced expression levels of tPAsequences in the presence of MTX, or by virtue of prior treatment oftransformed cells with MTX.

As described in U.S. Ser. No. 398,003, tPA is a fibrinolytic substancewhich can be recovered from human melanoma cells (EPO Patent ApplicationPubln. No. 0041766). This product has been isolated and characterizedWeiman et al, The Lancet, II (8250): 1018 (1981)!. Its fibrinolyticactivity is analogous to that of two commercially available proteins,streptokinase and urokinase, which are indicated for the treatment ofacute cardiovascular diseases such as myocardial infarct, stroke,pulmonary embolism, deep vein thrombosis, peripheral arterial occlusionand other venous thromboses. The etiological basis for these diseases isapparently either a partial or total occlusion of a blood vessel by ablood clot. Thus traditional anticoagulant therapy for example,treatment with heparin or coumarine, is not effective as it will merelyprevent the formation of further clots, but not result in thedissolution of clots already formed. The fibrinolytic agents,streptokinase, urokinase; and plasminogen activator all operatesimilarly. They convert the inactive precursor plasminogen into plasminwhich is capable of dissolving the fibrin of which these clots arecomposed. Plasminogen activator has a high affinity for fibrin, and thuspreferentially activates plasminogen associated with the fibrin desiredto be dissolved. On the other hand, streptokinase and urokinase do not;hence, much of the plasmin formed is formed in circulating blood and isneutralized before it can reach the targeted clot. Furthermore, as thesecompounds create circulating rather than fibrin bound plasmin, otherclotting factor proteins in circulation such as fibrinogen, Factor V,and Factor VIII are also attacked by the activated protein causing ahemorrhagic potential. Furthermore, streptokinase is stronglyimmunogenic.

Plasminogen activator overcomes the foregoing difficulties byspecifically attacking plasminogen already bound to fibrin. The presentinvention concerns a method of increasing and controlling the productionof this valuable protein in recombinant cultures by effecting control onamplification of the sequence for DHFR protein.

SUMMARY OF THE INVENTION

In one aspect, the invention herein concerns plasmids which containcoding sequences for human tissue plasminogen activator (tPA) and a DHFRprotein, and which are effective in expressing both of them. In anotheraspect, the invention concerns cells transformed with these vectors.

In other aspects, the invention also concerns methods for producing tPAby taking advantage of the environmentally controlled response of DHFRcoding sequences co-transfected with the tPA sequence, and the tPA soproduced.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram of the construction of the exemplifiedDHFR (mutant or wild type)/tPA encoding plasmids.

DETAILED DESCRIPTION A. Definitions

As used herein:

Human "tissue plasminogen activator" (tPA) is a fibrinolytic protein asdescribed in U.S. Ser. No. 272,093, filed Jun. 11, 1980, now abandoned,which is a continuation in part of Ser. No. 183,638 filed Sep. 3, 1980,now abandoned, both incorporated herein by reference.

"DHFR protein" refers to a protein which is capable of the activityassociated with dihydrofolate reductase (DHFR) and which, therefore, isrequired to be produced by cells which are capable of survival on mediumdeficient in hypoxanthine, glycine, and thymidine (-HGT medium). Ingeneral, cells lacking DHFR protein are incapable of growing on thismedium, cells which contain DHFR protein are successful in doing so.

"Cells sensitive to MTX" refers to cells which are incapable of growingon media which contain the DHFR inhibitor methotrexate (MTX). Thus,"cells sensitive to MTX" are cells which, unless genetically altered orotherwise supplemented, will fail to grow under ambient and mediumconditions suitable for the cell type when the MTX concentration is 0.2μg/ml or more. Some cells, such as bacteria, fail to exhibit MTXsensitivity due to their failure to permit MTX inside their cellboundaries, even though they contain DHFR which would otherwise besensitive to this drug. In general, cells which contain, as their DHFRprotein, wild type DHFR will be sensitive to methotrexate if they arepermeable or capable of uptake with respect to MTX.

"Wild type DHFR" refers to dihydrofolate reductase as is ordinarilyfound in the particular organism in question. Wild type DHFR isgenerally sensitive in vitro to low concentrations of methotrexate.

"DHFR protein with low binding affinity for MTX" has a functionaldefinition. This is a DHFR protein which, when generated within cells,will permit the growth of MTX sensitive cells in a medium containing 0.2μg/ml or more of MTX. It is recognized that such a functional definitiondepends on the facility with which the organism produces the "DHFRprotein with low binding affinity for MTX" as well as upon the proteinitself. However, as used in the context of this invention, such abalance between these two mechanisms should not be troublesome. Theinvention operates with respect to conferring the capability ofsurviving these levels of MTX, and it is not consequential whether theability to do so is impacted by increased expression in addition to theinnate nature of the DHFR produced.

"Expression vector" includes vectors which are capable of expressing DNAsequences contained therein, where such sequences are operably linked toother sequences capable of effecting their expression. It is implied,although not always explicitly stated, that these expression vectorsmust be replicable in the host organisms either as episomes or as anintegral part of the chromosomal DNA. Clearly a lack of replicabilitywould render them effectively inoperable. In sum, "expression vector" isgiven a functional definition, and any DNA sequence which is capable ofeffecting expression of a specified DNA code disposed therein isincluded in this term as it is applied to the specified sequence. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of "plasmids" which refer to circular double strandedDNA loops which, in their vector form are not bound to the chromosome.In the present specification, "plasmid" and "vector" are usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors which serve equivalent functions and which becomeknown in the art subsequently hereto.

"Recombinant host cells" refers to cells which have been transformedwith vectors constructed using recombinant DNA techniques. As definedherein, tPA produced in the amounts achieved by virtue of thistransformation, rather than in such lesser amounts, or, more commonly,in such less than detectable amounts, as would be produced by theuntransformed host.

B. Detailed Description

B.1 Host Cell Cultures and Vectors

The vectors and methods disclosed herein are suitable for use in hostcells over a wide range of prokaryotic and eukaryotic organisms.

In general, of course, prokaryotes are preferred for cloning of DNAsequences in constructing the vectors useful in the invention. Forexample, E. coli K12 strain 294 (ATCC No. 31446) is particularly useful.Other microbial strains which may be used include E. coli strains suchas E. coli B, and E. coli X1776 (ATCC No. 31537). These examples are, ofcourse, intended to be illustrative rather than limiting.

Prokaryotes may also be used for expression. The aforementioned strains,as well as E. coli W3110 (F⁻, λ⁻, prototrophic, ATTC No. 27325), bacillisuch as Bacillus subtilus, and other enterobacteriaceae such asSalmonella typhimurium or Serratia marcesans, and various pseudomonasspecies may be used.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR 322, a plasmid derived from an E. colispecies (Bolivar, et al., Gene 2: 95 (1977)). pBR322 contains genes forampicillin and tetracycline resistance and thus provides easy means foridentifying transformed cells. The pBR322 plasmid, or other microbialplasmid must also contain, or be modified to contain, promoters whichcan be used by the microbial organism for expression of its ownproteins. Those promoters most commonly used in recombinant DNAconstruction include the β-lactamase (penicillinase) and lactosepromoter systems (Chang et al, Nature, 275: 617 (1978); Itakura, et al,Science, 198: 1056 (1977); (Goeddel, et al Nature 281: 544 (1979)) and atryptophan (trp) promoter system (Goeddel, et al, Nucleic Acids Res., 8:4057 (1980); EPO Appl Publ No. 0036776). While these are the mostcommonly used, other microbial promoters have been discovered andutilized, and details concerning their nucleotide sequences have beenpublished, enabling a skilled worker to ligate them functionally withplasmid vectors (Siebenlist, et al, Cell 20: 269 (1980)).

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example, (Stinchcomb, et al,Nature, 282: 39 (1979); Kingsman et al, Gene, 7: 141 (1979); Tschemper,et al, Gene, 10: 157 (1980)) is commonly used. This plasmid alreadycontains the trp1 gene which provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, for exampleATCC No. 44076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)). The presenceof the trp1 lesion as a characteristic of the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem., 255: 2073(1980)) or other glycolytic enzymes (Hess, et al, J. Adv. Enzyme Reg.,7: 149 (1968); Holland, et al, Biochemistry, 17: 4900 (1978)), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3' of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization (Holland, ibid.). Anyplasmid vector containing yeast-compatible promoter, origin ofreplication and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)!. Examples of such useful host cell linesare VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, andW138, BHK, COS-7 and MDCK cell lines. Expression vectors for such cellsordinarily include (if necessary) an origin of replication, a promoterlocated in front of the gene to be expressed, along with any necessaryribosome binding sites, RNA splice sites, polyadenylation site, andtranscriptional terminator sequences. It will be understood that thisinvention, although described herein in terms of a preferred embodiment,should not be construed as limited to those sequences exemplified.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment which also contains the SV40 viral origin of replication(Fiers, et al, Nature, 273: 113 (1978) incorporated herein by reference.Smaller or larger SV40 fragments may also be used, provided there isincluded the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the viral origin of replication.Further, it is also possible, and often desirable, to utilize promoteror control sequences normally associated with the desired gene sequence,provide such control sequences are compatible with the host cellsystems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g. Polyoma, Adeno, VSV, BPV, etc.) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

B.2 Selection of Cell Lines

In selecting a preferred host cell for transfection by the vectors ofthe invention, it is appropriate to select the host according to thetype of DHFR protein employed. If wild type DHFR protein is employed, itis preferable to select a host cell which is deficient in DHFR, thuspermitting the use of the DHFR coding sequence as a marker forsuccessful transfection in selective medium which lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, Proc. Natl.Acad. Sci. (USA) 77: 4216 (1980), incorporated herein by reference.

On the other hand, if DHFR protein with low binding affinity for MTX isused as the controlling sequence, it is not necessary to use DHFRresistant cells. Because the mutant DHFR is resistant to methotrexate,MTX containing media can be used as a means of selection provided thatthe host cells themselves are methotrexate sensitive. Most eukaryoticcells which are capable of absorbing MTX appear to be methotrexatesensitive. One such useful cell line is a CHO line, CHO-K1 ATCC No. CCL61.

The example which is set forth hereinbelow describes use of CHO cells ashost cells, and expression vectors which include the SV40 origin ofreplication as a promoter. However, it would be well within the skill ofthe art to use analogous techniques to construct expression vectors forexpression of desired protein sequences in alternative eukaryotic hostcell cultures.

B.3 Methods Employed

If cells without formidable cell wall barriers are used as host cells,transfection is carried out by the calcium phosphate precipitationmethod as described by Graham and Van der Eb, Virology, 52: 546 (1978).However, other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used.

If prokaryotic cells or cells which contain substantial cell wallconstructions are used, the preferred method of transfection is calciumtreatment using calcium chloride as described by Cohen,. F. N. et alProc. Natl. Acad. Sci. (USA), 69: 2110 (1972).

Construction of suitable vectors containing the desired coding andcontrol sequences employ standard ligation techniques. Isolated plasmidsor DNA fragments are cleaved, tailored, and religated in the formdesired to form the plasmids required.

Cleavage is performed by treating with restriction enzyme (or enyzmes)in suitable buffer. In general, about 1 μg plasmid or DNA fragments isused with about 1 unit of enzyme in about 20 μl of buffer solution.(Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer.) Incubation times of about 1hour at 37° C. are workable. After incubations, protein is removed byextraction with phenol and chloroform, and the nucleic acid is recoveredfrom the aqueous fraction by precipitation with ethanol.

If blunt ends are required, the preparation is treated for 15 minutes at15° with 10 units of Polymerase I (Klenow), phenol-chloroform extracted,and ethanol precipitated.

Size separation of the cleaved fragments is performed using 6 percentpolyacrylamide gel described by Goeddel, D., et al, Nucleic Acids Res.,8: 4057 (1980) incorporated herein by reference.

For ligation approximately equimolar amounts of the desired components,suitably end tailored to provide correct matching are treated with about10 units T4 DNA ligase per 0.5 μg DNA. (When cleaved vectors are used ascomponents, it may be useful to prevent religation of the cleaved vectorby pretreatment with bacterial alkaline phosphatase.)

The ligation mixture was used to transform E. coli K12 strain 294 (ATCC31446), and successful transformants were selected by ampicillinresistance. Plasmids from the transformants were prepared, analyzed byrestriction and/or sequenced by the method of Messing, et al, NucleicAcids Res., 9:309 (1981) or by the method of Maxam, et al, Methods inEnzymology, 65:499 (1980).

Amplification of DHFR protein coding sequences is effected by growinghost cell cultures in the presence of approximately 20-500,000 nMconcentrations of methotrexate, a competitive inhibitor of DHFRactivity. The effective range of concentration is highly dependent, ofcourse, upon the nature of the DHFR gene, protein and thecharacteristics of the host. Clearly, generally defined upper and lowerlimits cannot be ascertained. Suitable concentrations of other folicacid analogs or other compounds which inhibit DHFR could also be used.MTX itself is, however, convenient, readily available and effective.

B.4 Results Obtainable

The methods of the invention permit the production in host cell culturesof antigenically active tPA protein in amounts greater than 0.1 pg percell per day. With suitable application of amplifying conditions,amounts greater than 20 pg can be obtained. Stated in alternate terms,gene expression levels resulting in production of more than 9×10⁻⁶units, or, with suitable amplification, more than 18×10⁻⁵ units of tPAactivity are achieved.

C. Examples

The following examples are intended to illustrate but not to limit theinvention. In the examples here, a CHO cell line suitable for the typeof DHFR protein coding sequence to be introduced was employed as a hostcell culture in each case. However, other eukaryotic and prokaryoticcells are suitable for the method of the invention as well.

C.1 Production of tPA Using DHFR Protein with a Low Binding Affinity forMTX

C.1.A Vector Construction

The sequence encoding human tissue plasminogen activator (tPA) isinserted, an expression plasmid for a mutant DHFR with low bindingaffinity for MTX, described in copending application U.S. Ser. No.06/459,151, incorporated herein by reference, by the following procedure(see FIG. 1):

cDNA plasmids encoding tPA have been described by Goeddel et al,application Ser. No. 374,860, filed May 5, 1982, and Ser. No. 398,003,filed 14 Jul. 1982, which hereby incorporated by reference. Humanmelanoma cells (Bowes) (ATCC CRL 9607) were used. The melanoma cellswere cultured to confluent monolayers in 100 ml Earles Minimal EssentialMedia supplemented with sodium bicarbonate (0.12 percent finalconcentration), 2 mM glutamine and 10 percent heat-inactivated fetalcalf serum. To confirm that the melanoma cells were actively producinghuman plasminogen activator, human melanoma cells were cultured toconfluency in a 24 well microtiter dish. Either in the presence orabsence of 0.33 μM in the protease inhibitor aprotinin, the cells werewashed once with phosphate buffered saline and 0.3 ml of serum freemethionine free medium was added. 75 μCi of ³⁵ S!-methionine was addedand the cells were labeled at 37° C. for 3 hours. At the end of the 3hour labelling period the media was removed from the cells and treatedwith either plasminogen activator specific IgG or pre-immune sera forimmunoprecipitation (Oppermann, et al., Virology 108, 47 (1981)). Theimmunoprecipitated products were displayed by electrophoresis on a 10percent SDS-acrylamide gel. The slab gel was fixed, dried and subjectedto fluorography.

Total RNA from melanoma cell cultures was extracted essentially asreported by Ward, et al., (J. Virol. 9, 61 (1972)). Cells were pelletedby centrifugation and then resuspended in 10 mM NaCl, 10 mM Tris-HCl pH7.5, 1.5 mM MgC1₂. Cells were lysed by the addition of NP-40 (1 percentfinal concentration), and nuclei were pelleted by centrifugation. Thesupernatant contained the total RNA which was further purified bymultiple phenol and chloroform extractions. The aqueous phase was made0.2M in NaCl and then total RNA was precipitated by the addition of twovolumes of ethanol. Oligo-dT cellulose chromatography was utilized topurify mRNA from the total RNA preparations. Typical yields from 10grams of cultured melanoma cells were 5 to 10 milligrams of total RNAand 50-200 micrograms of Poly(A) plus mRNA.

Fractionation of PolyA⁺ mRNA (200 μg) (Aviv, et al., Proc. Natl. Acad.Sci. (USA) 69, 1408 (1972)) was performed by electrophoresis throughurea-agarose gels. The slab agarose gel (Lehrach, et al., Biochemistry16, 4743 (1977) and Lynch, et al. Virolog. 98, 251 (1979)) was composedof 1.75 percent agarose, 0.025M sodium citrate, pH 3.8 and 6M urea.Electrophoresis was performed for 7 hours at 25 milliamps and 4° C. Thegel was then fractionated with a razor blade. The individual slices weremelted at 70° C. and extracted twice with phenol and once withchloroform. Fractions were then ethanol precipitated and subsequentlyassayed by in vitro translation in a rabbit reticulocyte lysate system,Bethesda Research Lab. (Lodish, Ann. Rev. of Biochem. 45, 40 (1976) andPelham, et al., Eur. J. Biochem. 43, 247 (1976)), supplemented with dogpancreas microsomes as follows: Translations were performed using 25 μCiof ³⁵ S!-methionine and 500 nanograms of each gel slice RNA in a finalvolume of 30 μl containing 25 mM HEPES, 48.3 potassium chloride, 10 mMcreatine phosphate. 19 amino acids at 50 mM each, 1.1 mM magnesiumchloride 16.6 mM EDTA, 0.16 dithiothreitol 8.3 mM hemin, 16.6 μg/mlcreatine kinase, 0.33 calcium chloride, 0.66 mM EGTA, 23.3 mM sodiumchloride.

Incubations were carried out at 30° C. for 90 minutes. Dog pancreasmicrosomal membranes prepared from rough microsomes using EDTA forremoval of the ribosomes (Blobel, et al., J. Cell Biology 67, 852(1975)) were treated with nuclease as described (Shields, et al., J.Biol. Chemistry 253, 3753 (1978)) and were present in the translationmixture at a final concentration of 7 A₂₆₀ units/ml. Translationproducts or immunoprecipitated translation products were analyzed byelectrophoresis on 10 percent polyacrylamide gels in the sodium dodecylsulfate as previously described (Laemmli, Nature 227, 680 (1970)). Theunstained slab gels were fixed, dried and subjected to fluorography(Bonner, et al., Eur. J. Biochem. 46, 83 (1974)).

The resulting translation products from each gel fraction wereimmunoprecipitated with rabbit anti-human plasminogen activator specificIgG. One major immunoprecipitated polypeptide band was observed in thetranslation of RNA fraction number 7 and 8 (migration of 21 to 24 S)having a molecular weight of approximately 63,000 daltons. This band wasnot observed when preimmune IgG was used for immunoprecipitation whichsuggested these polypeptides were plasminogen activator specific.

Five μg of gel fractionated mRNA (gel slice 7 mRNA) was used for thepreparation of double stranded cDNA by standard procedures (Goeddel, etal., Nature 287, 411 (1980), Goeddel, et al., Nature 281, 544 (1979),and Wickens, et al., J. Biol. Chem. 253, 2483 (1978)). The cDNA was sizefractionated on a 6 percent polyacrylamide gel. The cDNA greater than350 base pairs in length (125 ng) was electroeluted. 30 ng of cDNA wasextended with deoxy(C) residues using terminal deoxynucleotidyltransferase (Chang, et al., Nature 275, 617 (1978)) and annealed with300 ng of the plasmid pBR322 (Bolivar, et al., Gene 2, 95 (1977)) whichhad been similarly tailed with deoxy(G) residues at the Pst I site(Chang, et al., Nature 275, 617 (1978)). The annealed mixture was thentransformed into E. coli K12 strain 294 (ATCC No. 31446). Approximately4,600 transformants were obtained.

Purified human plasminogen activator was obtained according to theprocedure of disclosed references (European Patent Application Publn.No. 0041766 and Weimar, W., et al., The Lancet Volume II 8254, 1018(1981)).

The molecule was scanned in order to locate regions best suited formaking synthetic probes, as follows:

To make the proteins susceptible to digestion by trypsin it was reducedand carboxymethylated. A 2 mg sample of plasminogen activator was firstdialyzed against 0.01 percent Tween 80 in water. The lyophilized proteinwas then dissolved in 2.5 ml of Tris-HCl buffer (pH 8.6) and 8 molar inurea. The disulfide bonds were reduced by additions of 0.1 ml of,β-mercaptoethanol. This reaction was carried out under nitrogen for 2hours. The reduced disulfides were alkylated to the carboxymethylderivative by the addition of iodoacetic acid in 1N NaOH. After 20minutes the reaction was stopped by dialysis against 0.01 percent Tween80 in water.

The resulting lyophilized carboxymethylated protein was redissolved in 3ml of 0.1M sodium phosphate buffer (pH 7.5). Trypsin (TPCK) was added (1to 50 ratio) and digested at 37° C. Aliquots (0.1 ml) were taken at 3hours, 6 hours, and 12 hours. A second addition of trypsin was madeafter 12 hours. The reaction was stopped after 24 hours by freezing thesample until it could be injected on the HPLC. The progress of thedigestion was determined by SDS gels on the aliquots. All gels wereblank except for a faint band on the 3 hours aliquot. This indicatedthat the 24 hour digestion was complete and no large peptides remained.

An analytical amount (ca. 0.5 ml) was injected into a high resolutionAltex C-8 ultrasphere 5 μ column with two runs. A gradient ofacetonitrile was made gradual (1 percent to 5 percent in 5 min, 5percent to 35 percent in 100 min=, 35-50 percent in 30 min). In one ofthe two preparative runs, the eluant was monitored at two wavelengths(210 mn and 280 nm). The ratio of the two wavelength absorptions wasused to indicate the tryptophan containing peptides.

The peptide peaks most likely to contain tryptophan, or that werebelieved useful for other reasons, were sequenced first. This enabledthat determination of the sequence around most of the tryptophans. Aftersequencing about 25 of the best possible peptide peaks, all the sequencedata that could be aligned was pooled to obtain a preliminary model ofthe primary structure of plasminogen activator. From this data andmodel, several possible probes were located.

The colonies were individually inoculated into wells of microtiterplates containing LB (Miller, Experiments in Molecular Genetics, p.431-433 Cold Spring Harbor Lab., Cold Spring Harbor, New York (1972))+5μl/ml tetracycline and stored at -20° C. after addition of DMSO to 7percent. Two copies of the colony library were grown up onnitrocellulose filters and the DNA from each colony fixed to the filterby the Grunstein Hogness procedure (Grunstein, et al., Proc. Natl. Acad.Sci. (USA) 72, 3961 (1975)).

The ³² P-labeled-TC(A,G) CA(A,G)TA(C,T)TCCCA probe was prepared (fromthe synthetic oligomer) (W-E-Y-C-D) 14-mer pool of 8 radiolabeledsynthetic deoxyoligonucleotides (14-mers) TC(AG)CA(AG)TA(CT)TCCCA codingfor the known amino acid sequence: tryptophan-glutamicacid-tryosine-cysteine-aspartic acid (W-E-Y-C-D). Filters containing4,600 transformants were prehybridized for 2 hours at room temperaturein 50 mM sodium phosphate pH 6.8, 5× SSC (Blin, et al., Nucleic AcidResearch 3, 2303 (1976)), 150 μg/ml sonicated salmon sperm DNA, 5×Denhardt's solution (Denhardt, Biochem. Biophys. Res. Comm. 23, 641(1966)) 10 percent formamide and then hybridized with 50x 10⁶ counts perminute of the labelled probe in the same solution. After an overnightincubation at room temperature, the filters were washed 3 times at roomtemperature in 6× SSC, 0.1 percent SDS for 30 minutes, once in 2× SSCand then exposed to Kodak XR-5 x-ray film with DuPont Lightning Plusintensifying screens for 16 hours.

Plasmid DNA was isolated by a rapid method (Birnboim, et al., NucleicAcids Research 7, 1513 (1979)) from all colonies showing a positivehybridization reaction. The cDNA inserts from these clones were thensequenced after subcloning fragments into the M13 vector mp 7 (Messing,et al., Nucleic Acids Research 9, 309 (1981)) and by the Maxam Gilbertchemical procedure (Maxam, et al., Methods in Enzymol. 65, 499 (1980)).Filter number 25 showed the hybridization pattern of a positiveplasminogen activator clone. The cDNA insert in clone 25E10 wasdemonstrated to be the DNA coding for plasminogen activator by comparingits amino acid sequence with peptide sequence obtained from purifiedplasminogen activator and by its expression product produced in E. colias described in more detail infra. Clone 25E10 was 2304 base pairs inlength with the longest open reading frame encoding protein of 508 aminoacids (MW of 56,756) and containing a 772 bp 3' untranslated region.This cDNA clone lacked the N-terminal coding sequences.

50 μg of pPA25E10 (supra) were digested with Pst I and the 376 bpfragment isolated by electrophoresis on a 6 percent polyacrylamide gel.Approximately 3 μg of this fragment was isolated from the gel byelectroeluting, digested with 30 units of Dde I for 1 hour at 37° C.phenol and chloroform extracted, and ethanol precipitated. The resultingDde I sticky ends were extended to blunt ends by adding 5 units of DNApolymerase I (Klenow fragment) and 0.1 mM each of dATP, dCTP, dGTP, dTTPto the reaction mixture and incubating at 4° C. for 8 hours. Afterextraction with phenol and chloroform, the DNA was digested with 15units of Nar I for 2 hours and the reaction mixture electrophoresed on a6 percent polyacrylamide gel. Approximately 0.5 μg of the desired 125 bpblunt end Nar I fragment was recovered. This fragment codes for aminoacids number 69 through 110 of the mature full length plasminogenactivator protein.

For isolation of the 1645 bp Nar I-Bgl II fragment, 30 μg of pPA25E10were digested with 30 units of Nar I and 35 units of Bgl II for 2 hoursat 37° C. and the reaction mixture electrophoresed on a 6 percentpolyacrylamide gel. Approximately 6 μg of the desired 1645 bp Nar I-BglII fragment were recovered.

The plasmid pΔ4R1SRC is a derivative of the plasmid pSRCexl6 (McGrathand Levinson, Nature 295, 423 (1982)) in which the Eco RI sites proximalto the trp promoter and distal to the SRC gene have been removed byrepair with DNA polymerase I (Itakura, et al., Science 198, 1056(1977)), and the self-complementary oligodeoxynucleotide AATTATGMTTCAT(synthesized by the phosphotriester method (Crea, et al., Proc. Natl.Acad. Sci. (USA) 75, 5765 (1978))) was inserted into the remaining EcoRI site immediately adjacent to the Xba I site. 20 μg of pΔR1SRC weredigested to completion with Eco RI, phenol and chloroform extracted, andethanol precipitated. The plasmid was then digested with 100 units ofnuclease S1 at 16° C. for 30 minutes in 25 mM sodium acetate (pH 4.6), 1mM ZnC1₂ and 0.3M NaCl to create a blunt end with the sequence ATG.After phenol and chloroform extraction and ethanol precipitation, theDNA was digested with Bam HI, electrophoresed on a 6 percentpolyacrylamide gel, and the large (4,300 bp) vector fragment recoveredby electroelution.

The expression plasmid was assembled by ligating together 0.2 μg ofvector, 0.06 μg of the 125 bp blunt end-Nar I fragment and 0.6 μg of the1645 bp Nar I-Bgl II fragment with 10 units of T₄, DNA ligase for 7hours at room temperature and used to transform E. coli strain 294 (ATCCNo. 31446) to ampicillin resistance. Plasmid DNA was prepared from 26 ofthe colonies and digested with Xba I and Eco RI. Twelve of theseplasmids contained the desired 415 bp Xba I-Eco RI and 472 bp Eco RIfragments. DNA sequence analysis verified that several of these plasmidshad an ATG initiation codon correctly placed at the start of amino acidnumber 69 (serine). One of these plasmids, pΔRIPA° was tested andproduced the desired plasminogen activator.

0.4 μg of the synthetic oligonucleotide 5' TTCTGAGCACAGGGCG 3' was usedfor priming 7.5 μg of gel fraction number 8 (supra) to prepare doublestranded cDNA by standard procedures (Goeddel, et al., Nature 281, 544(1979), and Wickens, et al., J. Biol. Chem 253, 2483 (1978)). The cDNAwas size fractionated on a 6 percent polyacrylamide gel. A size fractiongreater than 300 base pairs (36 ng) was electroeluted. 5 ng cDNA wasextended with deoxy(C) residues using terminal deoxycytidyl transferase(Chang, et al., Nature 275, 617 (1978)) and annealed with 50 ng of theplasmid pBR322 (Bolivar, et al., Gene 2, 95 (1977)) which had beensimilarly tailed with deoxy(G) residues at the Pst I site (Chang, etal., sura). The annealed mixture was then transformed into E. coli K12strain 294. Approximately 1,500 transformants were obtained.

Since the cDNA priming reaction had been done using a synthetic fragmentthat hybridized 13 base pairs from the N-terminal of clone 25E10, noconvenient restriction fragment was available in this 29 base pairregion (which includes the 16-mer sequence) for screening the cDNAclones. Therefore, it was necessary to isolate a human plasminogenactivator genomic clone in order to identify any primer extending cDNAclones containing N-terminal plasminogen activator coding sequences.

The first step in this process involved establishing the fact that onlya single homologous plasminogen activator gene is present in humangenomic DNA. To determine this, a Southern hybridization was performed.In this procedure (Southern, J. Mol. Biol. 98, 503 (1975)), 5 μg of highmolecular weight human lymphocyte DNA (prepared as in Blin, et al.,Nucleic Acid Research 3, 2303 (1976)) was digested to completion withvarious restriction endonucleases, electrophoresed on 1.0 percentagarose gels (Lawn, et al., Science 212, 1159 (1981)) and blotted to anitrocellulose filter (Southern, supra). A ³² P-labelled DNA probe wasprepared (Lawn, et al., Cell 15 1157 (1978)) from the 5' end of the cDNAinsert of the cDNA clone 25E10 (a 230 bp Hpa II-Rsa I fragment) andhybridized (Fritsch, et al., Cell 19, 959 (1980) with the nitrocellulosefilter. 35×10⁶ counts per minute of the probe were hybridized for 40hours and then washed as described (Fritsch, et al. supra). Twoendonuclease digestion patterns provide only a single hybridizing DNAfragment: Bgl II (5.7 Kbp) and Pvu II (4.2 Kbp). Two hybridizing DNAfragments were observed with Hind II (5.1 Kbp and 4.3 Kbp). Takentogether, these data suggest the presence of only a single plasminogenactivator gene in the human genome, and that this gene contains at leastone intervening sequence.

The strategy used to identify λ phage recombinants carrying plasminogenactivator genes consisted in detecting nucleotide homology with aradioactive probe prepared from the plasminogen activator clone p25E10.One million recombinant λ phage were plated out on DP 50 Sup F at adensity of 10,000 pfu/15 cm plate, and nitrocellulose filter replicaswere prepared for each plate by the method of Benton and Davis (Benton,et al., Science 196, 180 (1 977)). A ³² P-labelled DNA probe wasprepared by standard procedures (Taylor, et al., Biochem. Biophys. Acta442, 324 (1976)) from a 230 base pair Hpa II-Rsa I fragment located 34base pairs from the 5' end of the clone p25E10. Each nitrocellulosefilter was prehybridized at 42° C. for 2 hours in 50 mM sodium phosphate(pH 6.5), 5× SSC (Southern, supra), 0.05 mg/ml sonicated salmon spermDNA, 5× Denhardt's solution (Denhardt, supra), 50 percent formamide andthen hybridized with 50×10⁶ counts per minute of the labelled probe inthe same solution containing 10 percent sodium dextran sulfate (Wahl, etal., Proc. Natl. Acad. Sci. (USA) 76, 3683 (1979)). After an overnightincubation at 42° C., the filters were washed 4 times at 50° C. in 0.2×SSC, 0.1 percent SDS for 30 minutes, once in 2× SSC at room temperatureand then exposed to Kodak XR-5 x-ray film with Dupont Cronexintensifying screens overnight. A total of 19 clones were obtained whichhybridized with the probe. Phage DNA was prepared as previouslydescribed (Davis, et al., Advanced Bacterial Genetics, Cold SpringHarbor Laboratory, New York (1980)) from 6 recombinants. λ Clone C wasselected for preparation of a Pvu II. fragment colony screening. 30 μgof DNA was digested with Pvu II for 1 hour at 37° C., andelectrophoresed on 1.0 percent agarose gels. A 4.2 Kilobase pairfragment p reviously shown to contain plasminogen activator sequenceswas electroeluted and purified. A ³² P-labelled probe was prepared bystandard procedure (Taylor, et al., supra) for colony hybridizations asdescribed infra.

The colonies were transferred from plates and grown on nitrocellulosefilters and the DNA from each colony fixed to the filter by theGrunstein-Hogness procedure (Grunstein, et al., supra). A ³² P-labelledprobe was made by calf-thymus priming (Taylor, et al., supra) a 4.2kilobase pair Pvu II fragment from an isolate plasminogen activator λgenomic clone. Filters containing the 1,500 transformants werehybridized with 112×10⁶ cpm of ³² P-genomic Pvu II fragment.Hybridization was for 16 hours using conditions described by Fritsch, etal. (supra). Filters were extensively washed and then exposed to KodakXR-5 x-ray film with Dupont Lightning Plus intensifying screens for16-48 hours. Eighteen colonies clearly hybridized with the genomicprobe. Plasmid DNA was isolated from each of these colonies and wasbound to nitrocellulose filters and hybridized with the ³² P-labelledsynthetic oligonucleotide (16-mer) used for the original primingreaction. Of the 18 clones, seven hybridized with the kinased 16-mer.Upon sequence analysis after subcloning fragments into the m13 vectormp⁷ (Messing, et al., supra), one clone (pPA17) was shown to contain thecorrect 5' N-terminal region of plasminogen activator, a signal leadersequence and an 84 bp 5' untranslated region. From the two clonespPA25E10 and pPA17 the complete nucleotide sequence and restrictionpattern of a full length plasminogen activator clone were determined.

A reconstruction of the entire coding sequence was possible employingcommon Hha I restriction endonuclease site shared by both partial clonesPA17 and 25E10. A 55 bp Sau3Al-Hhal restriction fragment correspondingto amino acids 5-23 was isolated from the plasmid pPA17. The Sau3Alrestriction site was located at codon four of the presumed mature codingsequence and was used to remove the signal peptide coding region. A 263bp Hhal-Narl fragment (coding for amino acids 24-110) was also isolatedfrom plasmid p25E10. Two synthetic deoxyoligonucleotides were designedwhich restore the codons for amino acids 1-4, incorporate an ATGtranslational initiation codon and create an EcoRI cohesive terminus.These three fragments were then ligated together to form a 338 bpfragment coding for amino acids 1-110. This fragment and a 1645 bpNarl-BgIII fragment from p25E10 were then ligated between the EcoRI andBgIIII sites of the plasmid pLelFAtrp103 (Gray, et al., Nature 295, 503(1982)) to give the expression plasmid pEPAtrp12. The cloned t-PA geneis transcribed under the control of a 300 bp fragment of the E. coli trpoperon which contains the trp promoter, operator, and the Shine-Dalgarnosequence of trp leader peptide but lacks the leader peptide ATGinitiation codon (Goeddel, et al., Nature 287, 411 (1980)). Threefragments from overlapping tPA plasmids, pPA25E10(ATCC 40401), andpPA17(ATCC 40402), and are ptPAtrp12 (ATCC 40404) were prepared asfollows: Plasmid pPA17 was digested with Dde I, filled in using KlenowDNA polymerase I, and subcut with Pst I; the approximately 200 bpfragment containing 5' terminal tPA sequence thus generated wasisolated. The second tPA fragment was obtained by digesting ptPAtrp12with Pst I and Nar I and isolating the approximately 310 bp fragment.The third tPA fragment was obtained by digesting pPA25E10 with Nar I andBgl II and isolating the approximately 1645 bp fragment which contains,in addition to much of the tPA coding region, some 3' non-translatedsequences. Bacterial clones E. coli (pPA25E10), E. coli (pPA17) and E.coli (pΔRIPA°) have been deposited with the American Type CultureCollection and accorded accession numbers 67587, 67586 and 67585,respectively.

Plasmid p342E which expresses HBV surface antigen (also referred to aspHBs348-E) has been described by Levinson et al, patent application Ser.No. 326,980, filed Dec. 3, 1981, which is incorporated herein byreference. The origin of SV40 was isolated by digesting SV40 DNA withHind III, and converting the Hind III ends to EcoRI ends by the additionof a converter (AGCTGAATTC). This DNA cut with PvuII, and RI linkersadded. Following digestion with EcoRI, the 348 base pair fragmentspanning the origin was isolated by polyacrylamide gel electrophoresisand electroelution, and cloned in pBR322. Expression plasmids pHBs348-Eand pHBs348-L were constructed by cloning the 1986 base-pair fragmentresulting form EcoRI and BgIII digestion of HBV (Animal Virus Genetics(Ed. Fields, Jaenisch and Fox), Chapter 5, p. 57, Academic Press, NewYork (1980)) (which spans the gene encoding HBsAg) into the plasmid pML(Lusky and Botchan, Nature 293, 79 (1981)) at the EcoRI and BamHI sites.(pML is a derivative of pBR332 which has a deletion eliminatingsequences which are inhibitory to plasmid replication in monkey cells(Lusky and Botchan,--supra.)) The resulting plasmid (pRI-BgI) was thenlinearized with EcoRI, and the 348 base-pair fragment representing theSV40 origin region was introduced into the EcoRI site of pRI-BgI. Theorigin fragment can insert in either orientation. Since this fragmentencodes both the early and late SV40 promoters in addition to the originof replication, HBV genes could be expressed under the control of eitherpromoter depending on this orientation (pHBS348-E representing HBs undercontrol of the early promoter). pE342 is modified by digesting withtrace amounts of Eco RI, filling in the cleaved site using Klenow DNAploymerase I, and ligating the plasmid back together, thus removing theEco RI site preceding the SV40 origin in pE342. The resulting plasmid,designated pE342ΔR1, is digested with Eco RI, filled in using Klenow DNApolymerase I, and subcut with Bam HI. After electrophoresing onacrylamide gel, the approximately 3500 bp fragment is electroeluted,phenol-chloroformed, and ethanoled as above.

The thus prepared p342E 3500 bp vector, and above described tPAfragments comprising approximately 2160 bp were ligated together usingstandard techniques. A plasmid containing the three tPA encodingfragments in the proper orientation was isolated, characterized, anddesignated pE342-tPA. This plasmid was digested with Sac II and treatedwith bacterial alkaline phosphatase (BRL). To provide the DHFR sequence(along with control sequences for its expression) an approximately 1700bp fragment was generated by SacII digestion of pEHER. (pEHER is aplasmid expressing mutant DHFR described in copending U.S. Ser. No.06/459,151.) This fragment was ligated into the pE342-tPA plasmid tocreate pETPAER400, a plasmid which is analagous to pEHER except that theHBsAg coding region has been replaced by the cDNA sequences from tPA.

C.1.B Expression and Amplification of the tPA Sequence

pETPAER400 (pETPER) was transfected into both dhfr⁻ (CHO-DUX B11)obtained by permission from Urlaub and Chasin, and DHFR⁺ CHO-K1 (ATCCCCL61) cells by the method of Graham and Van der Eb (supra). Transformeddhfr⁻ cells were selected by growth in glycine, hypoxanthine andthymidine deficient medium. Transformed DHFR⁻ cells were selected bygrowth in ≧100 nM MTX. Colonies which arose on the appropriate selectionmedium were isolated using cloning rings and propagated in the samemedium to several generations.

For amplification cells from the colonies are split into mediacontaining 5×10⁴, 10⁵, 2.5×10⁵, 5×10⁵, and 10⁶ nM MTX and passagedseveral times. Cells are plated at very low (10² -10³ cells/plate) celldensities in 10 cm dishes and the resulting colonies are isolated asusual.

C.1.C Assay Methods

Expression of tPA in the transfected amplified colonies may convenientlybe assayed by the methods set forth in U.S. application 398,003.Briefly, for quantitative assay, the medium or extract to be tested isplaced in a solution containing plasminogen, and the amount of plasminformed is measured by monitoring the cleavage of a chromogenic substratesuch as S2251, Kabi Group Inc., Greenwich, Conn. An aliquot of thesample is mixed with 0.1 ml of 0.7 mg/ml plasminogen (in 0.5M Tris-HCl,pH 7.4, containing 0.012M NaCl) and the volume adjusted to 0.15 ml. Themixture is incubated at 37° C. for ten minutes, 0.35 ml of S2251 (1.0 nMsolution in the above buffer) is added and the reaction continued for 30minutes at 37° C. Acetic acid (25 μl) is added to terminate thereaction. The samples are centrifuged and the absorbance at 405 nm ismeasured. Quantitation of the amount of activity is obtained bycomparison with a standard urokinase solution. The assay conditions fordetection of a full length plasminogen activator were modified by theaddition of fibrinogen (0.2 mg) to the solution. Fibrinogen results in astimulation of the activity of plasminogen activator observed, thereforeresulting in somewhat elevated levels of activity. Activity was recordedin Plough units, wherein 90,000 Plough units is equal to the activityexhibited by 1 mg of purified tissue plaminogen activator.

Coamplification of DHFR and tPA sequences is assayed by isolating DNAfrom confluent monolayers of amplified colonies as follows: Confluentmonolayers in 150 mm plates are washed with 50 ml sterile PBS and lysedby the addition of 5 ml of 0.1 percent SDS, 0.4M CaCl₂, 0.1M EDTA, pH 8.After 5-10 minutes, the mixture is removed, phenol extracted, chloroformextracted, and ethanol precipitated. The DNA is resuspended in 1 ml (per150 mm plate) 10 mM Tris pH 8, 1 mM EDTA (TE), RNase added to 0.1 mg/ml,and the solution incubated 30 minutes at 37°. SDS is then added to 0.1percent and pronase (Sigma) is added to 0.5 mg/ml. After 3-16 hoursincubation at 37°, the solution is again phenol extracted, chloroformextracted, and ethanol precipitated as usual. The DNA pellet isresuspended in 0.5 ml water and digested with restriction enzymes as perthe standard protocol. Approximately 5-10 μg of digested DNA iselectrophoresed in an agarose gel 1 percent agarose in Tris-acetatebuffer (40 mM Tris, 1 mM EDTA, made to pH 8.2 with acetic acid)!;Crouse, et al, J. Biol. Chem., 257: 7887 (1982)). After bromphienol bluedye had migrated 2/3 of the way down the gel, the gel is removed andstained with ethidium bromide. After visualizing the DNA withultraviolet light, the gel is treated with HCl, NaOH, and NaCl-Tris andtransferred to nitrocellulose filters according to the procedure ofSouthern (J. Mol. Biol. 98: 503. (1975)). The filters are thenhybridized with a nick translated probe made from the 1700 bp SacIIfragment of pEHER (prepared and hybridized as described above), or fromthe approximately 1970 bp Bgl II fragment of pETPER.

C.2. Production of tPA in Conjunction with Wild Type DHFR Protein

C.2.A. Vector Construction

In a manner exactly analogous to that used in the construction ofpETPER, a plasmid containing the DNA sequence encoding wild type DHFR,pETPFR (ATCC 40403), was constructed. The construction was exactly asdescribed in Example C.1.A except that in place of plasmid pEHER as asource for the DHFR protein gene sequence, the plasmidpE342.HBV.E400.D22 described in copending U.S. Ser. No. 06/459,152 wassubstituted. pE342 is modified by partially digesting with EcoRI,filling in the cleaved site using Klenow DNA polymerase I, and ligatingthe plasmid back together, thus removing the EcoRI site preceding theSV40 origin in pE342. The resulting plasmid, designated pE342ΔR1, isdigested with EcoRI, filled in using Klenow DNA polymerase I, and subcutwith BamHI. After electrophoresing on acrylamide gel, the approximately3500 bp fragment is electroeluted, phenolchloroform extracted, andethanol precipitated as above. The 5' nontranslated leader region ofHBsAg was removed by treatment with EcoRI and with Xba, and theanalogous 150 bp EcoRI-Xba fragment of a hepatitis expression plasmidpHS94 (Liu, et al., DNA 1, 213 (1982)) was inserted in its place tocreate pE342.HS94.HBV.

The 1600 bp Pst I insert of the DHFR cDNA plasmid DHFR-11 (Nunberg, etal., Cell 19, 355 (1980)) was treated with the exonuclease Bal31 inorder to remove the poly G:C region adjacent to the Pst I sites,digested with BgIII and the resulting fragments of approximately 660 bpisolated from gels. The Bal31-BgIII digested cDNA was ligated into apBR322 plasmid derivative containing a BgIII site. (Following digestionof pBR322 with Hind III, the plasmid fragment was filled in using KlenowDNA polymerase in the presence of the four deoxynucleotidetriphosphates, and subcut with BgIII.) The resulting plasmid, pDHFR-D22,has an EcoRI site situated 29 bp upstream of the fusion site betweenpBR322 and the 5' end of the DHFR cDNA. The EcoRI-BgIII fragmentencompassing the coding sequences of the cDNA insert was then excisedfrom pDHFR-D22 and ligated to EcoRI-BamHI digested pE342.HS94.HBV,creating the DHFR expression plasmid pE342.D22. pE342.HBV.D22 wasconstructed by ligating the EcoRI-TaqI fragment of cloned HBV DNA (Liu,et al., supra), to EcoRI-ClaI digested pE342.D22. This plasmid wasfurther modified by fusing an additional SV40 early promoter between theBgIII site and the ClaI site of the DHFR insert of pE342.HBV.D22,creating pE342.HBV.E400.D22.. The plasmid pE342.HBV.E400.D22 is exactlythe same as pEHER except for a single base pair difference between wildtype and mutant DHFR. Thus the resulting plasmid pETPFR is analogous inevery way to pETPER except that the DNA sequence encoding for wild typeDHFR is substituted for that of the mutant.

C.2.B Expression of tPA sequence

pETPFR was used to transfect DHFR deficient CHO cells (Urlaub and Chasin(supra)) using the calcium phosphate precipitation method of Graham andVan der Eb. Twenty-one colonies which arose on the selective medium(-HGT) were assayed by detection of plasmin formation as assessed by thedigestion of fibrin in an agar plate containing fibrin and plasminogen,described by Granelli-Piperno, et al, J. Exp. Med., 148: 223 (1978).

Four of the best positive clones were then assayed quantitatively forplasmin formation on a per cell basis according to the method set forthin C.1.C.

Upon such quantitative determination it was found that the four clonestested exhibited the same or comparable tPA secretion into the medium,determined as units/cell/day. Subclones were prepared by transferringinocula from two of the clones into separate plates containing -HGTmedium. Two of the resulting subclones, 18B and 1 were used for furtheranalysis.

C.2.C. Amplification and tPA Production Levels

The above subclones were plated at 2×10⁵ cells per 100 mm plates in 50nM MTX to promote amplification. Those cells which survived, whenassayed as described above, gave, in all cases, about 10 times theunamplified amount of plasminogen activator activity. Two of theseclones were chosen for further study and were named 1-15 and 18B-9.

Subclone 1-15 was further amplified by seeding 2×10⁵ cells in 100 mmplates containing 500 nM MTX. Assay of the cells thus amplified yieldeda further increase (of about 3 fold) in tPA production; when assayedquantitatively by the method of C.1.C, levels were in the range of7×10⁻⁴ units/cell/day. A portion of these amplified cells was thentransferred and maintained in the presence of 10,000 nM MTX. Subclonesof 1-15, and 18B-9 were further tested after being maintained forapproximately 1-2 months at the conditions specified in Table 1.

                  TABLE 1                                                         ______________________________________                                        Cell Line Growth Conditions                                                                             ng tPA/cell/day*                                    ______________________________________                                        1-15.sub.500                                                                            500 nM MTX      28.5 × 10.sup.-3                              1-15.sub.500                                                                            500 nM MTX      26.0 × 10.sup.-3                              1-15.sub.500                                                                            (-HGT medium, no MTX)                                                                          8.3 × 10.sup.-3                              1-15.sub.500                                                                            (-HGT medium, no MTX)                                                                         18.0 × 10.sup.-3                              1-15.sub.10,000                                                                         10 μM MTX    29.3 × 10.sup.-3                              1-15.sub.10,000                                                                         10 μM MTX    49.0 × 10.sup.-3                              18B-9     50 nM MTX       14.3 × 10.sup.-3                              18B-9     50 nM MTX       14.4 × 10.sup.-3                              18B-9     (-HGT medium, no MTX)                                                                         14.3 × 10.sup.-3                              18B-9     (-HGT medium, no MTX)                                                                         14.4 × 10.sup.-3                              1         (-HGT medium, no MTX)                                                                          1.0 × 10.sup.-3                              1         (-HGT medium, no MTX)                                                                          0.7 × 10.sup.-3                              ______________________________________                                         *tPA in the culture medium was assayed quantitatively in a                    radioimmunoassay as follows: Purified tPA and purified iodinated tracer       tPA derived from melanoma cells were diluted serially to include              concentration of 12.5 to 400 ng/ml in a buffer containing phosphate           buffered saline, pH 7.3, 0.5 percent bovine serum albumin, 0.01 percent       Tween 80, and 0.02 percent NaN3. Appropriate dilutions of medium samples      to be assayed were added to the radioactively labelled tracer proteins.       The antigens were allowed to incubate overnight at room temperature in th     presence of a 1:10,000 dilution of the IgG fraction of a rabbit antitPA       antiserum. Antibodyantigen complex was precipitated by absorption to goat     antirabbit IgG Immunobeads (BioRad) for two hours at room temperature. Th     beads were cleared by the addition of saline diluent followed by              centrifugation for ten minutes at 2000 × g at 4° Celsius.        Supernatants were discarded and the radioactivity in the precipitates was     monitored. Concentrations were assigned by comparison with the reference      standard.                                                                

The cell lines are as follows: Cell line "1" is an unamplified clonefrom the original set of four. "1-15₅₀₀ " is an amplified subclone ofcell line "1" which was amplified initially in 50 nM MTX to give 1-15and then transferred for further amplification into 500 nM MTX.1-15₁₀,000 is subclone of 1-15₅₀₀ which has been further amplified inthe presence of 10,000 nM MTX. Cell line 18B-9 is a subclone of one ofthe original four detected which had been amplified on 50 nM MTX. TheCHO cell line CHO 1-15₅₀₀ has been deposited with the American TypeCulture Collection and accorded accession number CRL 9606.

All of the amplified cells show increased levels of TPA production overthat exhibited by the unamplified cell culture. Even the unamplifiedculture produces amounts of tPA greater than 0.5 pg/cell/day;amplification results in levels approaching 50 pg/cell/day.

We claim:
 1. An expression vector which comprises:a first DNA sequenceencoding a DHFR protein; and a second DNA sequence encoding human tPA;wherein each of said first and second sequences is operably linked to aDNA sequence capable of effecting its expression in a CHO cell linetransformed with said vector.